Synthesis and biological evaluation of new hydrazide-Schiff bases

Abstract

A new series of N-({5-(substituted aryl)-furan-2-yl}-methylidene)-hydrazides were synthesized with a new class of Schiff bases derived from the reaction of substituted phenyl-1-ketohydrazide 2 or 2-(4-chloro-3-methylaryloxy) acetohydrazide 3 with different 5-(substituted aryl)-2-furfuraldehyde (1a-k) to yield substituted N-({5-(substituted aryl)-furan-2-yl}-methylidene)-hydrazides-Schiff bases (4a-f, 4g-k). The title compounds were subjected to in vitro antibacterial screening against Gram positive bacterial strains- S. aureus, B. cereus, E. faecalis and S. epidermidis, and Gram negative bacteria strains- E. coli, S. typhi, S. dysenteriae and K. pneumoniae. The synthesized Schiff bases were also evaluated for their anthelmintic activity against two species of earthworms (Pheretima posthuma and Perionyx excavatus). Some compounds have shown promising antibacterial and anthelmintic activities.

Introduction

Resistance to a number of antimicrobial agents by a variety of pathogenic bacteria is becoming a major global problem. The widespread use and misuse of antibiotics is one of the cause attributed to the emergence of drug resistance to majority of antibacterial agents (Mashrai et al., 2014). It warrants the scientific community to develop new antimicrobial agents with potent and broad spectrum of antimicrobial action against resistant pathogens vis a vis cheap and safe. Therefore, there is a wide scope on research on newer antibacterial agents. Another important public health problem is helminthiasis or worm infestations. It is a cause of several related diseases caused by a variety of worms and exists worldwide. Very few drugs are available in the market to treat all worm infestations. The situation is worsened due to unavailability of an ideal anthelmintic vaccine (Newton et al., 1999). Though research is going on but its delayed development has necessitated the discovery of new anthelmintic compounds that could be used effectively to circumvent the current situation.

Extensive literature survey shows that phenolic and haloaryl rings are associated with anthelmintic and anti-intestinal nematode (Duan et al., 2011), antioxidant (Valantina et al., 2009) and antibacterial activity (Husain et al., 2009). Schiff bases are important class of organic compounds with imine or azomethine (–C=N–) functional group. These are prepared by condensing primary amines with carbonyl compounds (Dhar and Taploo, 1982). Schiff bases are reported to possess diverse biological and pharmacological actions including potential anti-inflammatory (Gurupadayaya et al., 2008), antibacterial (Hearn et al., 2009), antitubercular (Aboul-Fadl et al., 2010) antiviral (Kumar et al., 2010.), anticonvulsant (Firke et al., 2009), and anthelmintic (Sharma et al., 2009) activities. It was proposed wide spectrum of biological activities of Schiff bases could be because of the interaction of nitrogen atom of azomethine with the active centers of cell constituents by forming a hydrogen bond and thus it interferes in normal cell processes (Venugopala and Jayashree, 2003).

Owing to the antimicrobial and anthelmintic activities of substituted furfuraldehyde, phenolic and haloaryl moiety, it was thought worthwhile to synthesize some new compounds comprising of these moieties as a part of their structures, with an objective to obtain potential antimicrobial and anthelmintic agents with enhanced biological activities. Therefore, several N-({5-(substitu-ted aryl)-furan-2-yl}-methylidene)-hydrazides (4a-f, 4g-k) Schiff bases were synthesized via synthesis of substituted phenyl-1-ketohydrazide 2 and 2-(4-chloro-2-methylaryloxy)-acetohydrazide 3 with different aromatic 5-(substituted aryl)-2-furfuraldehyde (1a-k). The novel Schiff bases were chemically characterized and screened for their antimicrobial and anthelmintic activities.

Materials and Methods

Chemistry

Melting points were determined in one end open capillary tubes and are uncorrected. Thin-layer chromatography was carried out to monitor the reactions using silica gel G plates. The IR spectra were recorded on Bruker, alpha E ATR FTIR spectrophotometer. ¹H-NMR spectra were recorded on Bruker spectrospin DPX-300 MHz in DMSO-d6; chemical shift (δ) values are reported in parts per million (ppm). The splitting pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were scanned on Brukers micrOTOF-QII, ESI mass spectro-photometer. Elemental analyses (C, H, and N) were done on a CHN rapid analyzer and reported in percentage abundance within ± 0.04% of the theoretical values. Spectral and micro-analysis data are consistent with the assigned structures.

Ketohydrazides (2), and 5-(phenyl substituted)-2-furfu-raldehydes (3) were prepared by reported method (Varshney et al., 2014).

General procedure for the synthesis of N-[{5-(substituted aryl)-furan-2-yl}-methylidene]-hydrazides (4a-f, 4g-k) Schiff bases: A mixture of ketohydrazide 2 (0.1 mol) and 5-(phenyl substituted)-2-furfuraldehyde 3 (0.05 mol) was refluxed on water bath for 7-8 hours in ethanol as solvent and in the presence of few drops of sulfuric acid as catalyst. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was allowed to cool; crystals formed were washed, dried and finally recrystallized with ethanol to furnish the desired compound (4a-f, 4g-k; Table I).

2-(2-Hydroxyphenyl)-N-[{5-(4-nitrophenyl)-furan-2-yl}-methylidene]-ketohydrazide (4a): IR (ν_max, cm^-1): 1653 (CO of CONH), 1619, 3331 (NH of CONH), 1552, 1456, 1218, 1192, 1069, 913, 729 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 4.51 (1H, s, OH), 6.73-7.80 (10H, m, Ar), 9.48 (1H, s, N=CH), 9.87 (1H, s, CONH); EI-MS (m/z, %); 351 [M+1, 100]; Anal. Calcd. for C₁₈H₁₃N₃O₅: C, 61.54; H, 3.73, N, 11.75. Found: C, 61.49; H, 3.74, N, 11.76.

2-(2-Hydroxyphenyl)-N-[5-(4-chlorophenyl)-furan-2-yl}-methylidene]-ketohydrazide (4b): IR (ν_max, cm^-1): 1651 (CO of CONH), 1623, 3312 (NH of CONH), 1548, 1459, 1216, 1158, 1064, 986, 718 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 4.22 (1H, s, OH), 6.82-7.69 (10H, m, Ar), 9.93 (1H, s, CONH), 10.12 (1H, s, N=CH);

EI-MS (m/z, %): 340 [M+1, 100]; Anal. Calcd. for C₁₈H₁₃ClN₂O₃: C, 63.54; H, 3.85, N, 10.40. Found: C, 63.58; H, 3.86, N, 10.44.

2-(2-Hydroxyphenyl)-N-({5-(4-bromophenyl)-furan-2-yl}-methylidene]-ketohydrazide (4c): IR (ν_max, cm^-1): 1644 (CO of CONH), 1604, 3328 (NH of CONH), 1546, 1449, 1228, 1151, 1022, 931, 737 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 4.04 (1H, s, OH), 6.73-7.82 (10H, m, Ar), 9.69 (1H, s, N=CH), 9.77 (1H, s, CONH); EI-MS (m/z, %): 385 [M+1, 100]; Anal. Calcd. for C₁₈H₁₃BrN₂O₃: C, 56.12; H, 3.40, N, 7.27. Found: C, 55.96; H, 3.43, N, 7.25.

2-(2-Hydroxyphenyl)-N-[{5-(4-methylphenyl)-furan-2-yl}-methylidene]-ketohydrazide (4d): IR (ν_max, cm^-1): 1635 (CO of CONH), 1633, 3342 (NH of CONH), 1542, 1458, 1237, 1164, 1075, 956, 749 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 3.16 (3H, s, CH₃), 4.33 (1H, s, OH), 6.57-7.82 (10H, m, Ar), 9.59 (1H, s, N=CH), 9.90 (1H, s, CONH); EI-MS (m/z, %): 320 (M+1, 100); Anal. Calcd. for C₁₉H₁₆N₂O₃: C, 71.24; H, 5.03, N, 8.04. Found: C, 71.25; H, 5.05, N, 8.07.

2-(2-Hydroxyphenyl)-N-[{5-(4-methoxyphenyl)-furan-2-yl}-methylidene]-ketohydrazide (4e): IR (ν_max, cm^-1): 1648 (CO of CONH), 1609, 3316 (NH of CONH), 1282 (C-O-C of Ar-OCH₃), 1540, 1424, 1227, 1167, 1054, 940, 745 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 4.23 (1H, s, OH), 6.87-7.79 (10H, m, Ar), 9.94 (1H, s, N=CH), 10.24 (1H, s, CONH); EI-MS (m/z, %): 336 (M+1, 100); Anal. Calcd. for C₁₉H₁₆N₂O₄: C, 67.85; H, 4.79, N, 8.33. Found: C, 67.82; H, 4.74, N, 8.29.

2-(2-Hydroxyphenyl)-N-[{5-(2,4-dinitrophenyl)-furan-2-yl}-methylidene]-ketohydrazide (4f): IR (ν_max, cm^-1): 1660 (CO of CONH), 1541, 3389 (NH of CONH), 1515, 1457, 1236, 1108, 1067, 992, 746 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 3.98 (1H, s, OH), 6.91-8.89 (9H, m, Ar), 9.86 (1H, s, N=CH), 9.95 (1H, s, CONH); EI-MS (m/z, %): 396 [M+1, 100]; Anal. Calcd. for C₁₈H₁₂N₄O₇: C, 54.55; H, 3.05, N, 14.14. Found: C, 54.24; H, 3.09, N, 13.90.

2-(4-Chloro-2-methylphenoxy)-N-[{5-(4-sulfoxyphenyl)-furan-2-yl}-methylidene]-acetohydrazide (4g): IR (ν_max, cm^-1): 1173 (C-O-C), 1672 (CO of CONH), 1626, 3353 (NH of CONH), 1547, 1464, 1265, 1169, 1102, 941, 742 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 2.24 ( 3H, s, CH₃), 4.61 (2H,s, OCH₂), 6.72-8.38 (9H, m, Ar-H), 9.42 (1H, s, N=CH), 10.18 (1H, s, CONH); EI-MS (m/z, %): 449 [M+1, 100]; Anal. Calcd. for C₂₀H₁₇ClN₂O₆S: C, 53.51; H, 3.82, N, 6.24. Found: C, 53.54; H, 3.85, N, 6.27.

2-(4-Chloro-2-methylphenoxy)-N-[{5-(2-carboxyphenyl)-furan-2-yl}-methylidene]-acetohydrazide (4h): IR (ν_max, cm^-1): 1167 (C-O-C), 1678 (CO of CONH), 1648, 3324 (NH of CONH), 1543, 1424, 1209, 1162, 1098, 875, 807, 775 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 2.29 ( 3H, s, CH₃), 4.65 (2H, s, OCH₂), 6.59-8.06 (9H, m, Ar-H), 9.54 (1H, s, N=CH), 10.16 (1H, s, CONH); EI-MS (m/z, %): 413 [M+1, 100]; Anal. Calcd. for C₂₁H₁₇ClN₂O₅: C, 61.01; H, 4.15, N, 6.79. Found: C, 61.35; H, 3.87, N, 6.82.

2-(4-Chloro-2-methylphenoxy)-N-[{5-(3-chlorophenyl)-furan-2-yl}-methylidene]-acetohydrazide (4i): IR (ν_max, cm^-1): 1175 (C-O-C), 1678 (CO of CONH), 1625, 3326 (NH of CONH), 1547, 1461, 1262, 1161, 1117, 865, 730 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 2.27 (3H, s, CH₃), 4.67 (2H, s, OCH₂), 6.67-8.48 (9H, m, Ar-H), 9.57 (1H, s, N=CH), 10.13 (1H, s, CONH); EI-MS (m/z, %): 404 [M+1, 100]; Anal. Calcd. for C₂₀H₁₆Cl₂N₂O₃: C, 59.57; H, 4.00, N, 6.95. Found: C, 59.63; H, 4.05, N, 6.96.

2-(4-Chloro-2-methylphenoxy)-N-[{5-(4-carboxyphenyl)-furan-2-yl}-methylidene]-acetohydrazide (4j): IR (ν_max, cm^-1): 1150 (C-O-C), 1675 (CO of CONH), 1623, 3324 (NH of CONH), 1542, 1462, 1264, 1167, 1121, 858, 720 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 2.29 ( 3H, s, CH₃), 4.61 (2H, s, OCH₂), 6.65-7.95 (9H, m, Ar-H), 9.36 (1H, s, N=CH), 10.19 (1H, s, CONH); EI-MS (m/z, %): 413 [M+1, 100]; Anal. Calcd. for C₂₁H₁₇ClN₂O₅: C, 61.01; H, 4.15, N, 6.79. Found: C, 61.28; H, 3.77, N, 6.82.

2-(4-Chloro-2-methylphenoxy)-N-[{5-(4-sulfacetamido-phenyl)-furan-2-yl}-methylidene]-acetohydrazide (4k): IR (ν_max, cm-1): 1158 (C-O-C), 1674 (CO of CONH), 1620, 3344 (NH of CONH), 1171 (SO₂ of SO₂NH), 1541, 1462, 1264, 1170, 1117, 852, 711 (C=C and C-H of aromatic ring); ¹H NMR: (DMSO-d₆, δ, ppm): 2.31 (3H, s, CH₃), 4.67 (2H, s, OCH₂), 6.71-7.95 (9H, m, Ar-H), 8.16 (1H, s, SO₂NH ), 9.32 (1H, s, N=CH), 10.21 (1H, s, CONH); EI-MS (m/z, %): 490 [M+1, 100]; Anal. Calcd. for C₂₂H₂₀ClN₃O₆S: C, 52.23; H, 3.98, N, 8.31. Found: C, 52.05; H, 4.02, N, 8.12.

Biological evaluation

The title compounds were evaluated for their antibacterial and anthelmintic activities.

Antibacterial activity

Antibacterial activity of the newly synthesized Schiff bases was evaluated by disc diffusion method (Cruickshank et al., 1975; Elmer et al., 2002) against four gram positive bacterial strains; S. aureus, B. cereus, E. faecalis, S. epidermidis, and four gram negative bacterial strains; E. coli, S. typhi, S. dysenteriae and K. pneumoniae taking ampicillin (20 µg/disc) as reference drug. Standard inoculums (1 mL/100 mL of medium) with suspension were introduced onto the surface of sterile agar plates, and a sterile bent glass spreader was used for even distribution of the inoculums. The discs mea-suring 6 mm in diameter were prepared from Whatman (Grade No. 1) filter paper and sterilized by dry heat for 1 hour. Three discs of test samples were placed on three portion together with one disc having the reference drug (ampicillin), and one disc impregnated with solvent (DMF) as negative control. The sterile discs previously soaked in a known concentration (25 µg/mL in dimethyl formamide) of the test compounds were placed in nutrient agar medium. Plates were inverted and incubated for 24 hours at 37 ± 1ºC. Diameters of zone of inhibition (mm) were determined and average diameter of test samples were calculated in triplicate sets. Zone of inhibition of test compounds were compared with that of the standard.

Anthelmintic activity

Anthelmintic activity was carried out against two different species of worms; Pheretima posthuma and Perionyx excavatus, at a 2 mg/mL concentration (Dahiya and Pathak 2007). Collected earthworms were washed with normal saline water to remove soil and fecal matter. Suspensions of samples were prepared by triturating synthesized compounds (100 mg) with Tween 80 (0.5%) and normal saline solution and the resulting mixtures were stirred for 30 min. The suspensions were diluted to obtain conc. of 0.2% w/v of the test samples. Suspension of reference drug albendazole (0.2% w/v) was prepared with the same manner. Three sets of five earthworms of almost similar sizes (approx. 2 inch in length) were placed in petri plates of 4 inch diameter containing 50 mL of suspension of test samples and reference drug. Another set of five earthworms was kept as control in 50 mL suspension of distilled water and tween 80 (0.5%). The paralyzing and death times were noted and their mean was calculated for triplicate sets.

Statistical analysis

The statistics i.e. one way ANOVA and t-test were applied on the values of mean ± SEM of triplicates (n=3) zone of growth of inhibition of test compounds, and compared with standard drug ampicillin with control as DMF (dimethyl formamide). While data of anthelmintic activity of the test compounds was analyzed by mean ± SD (n=5) and compared with albendazole.

Results and Discussion

Chemistry

The primary aim of this study was to condense a biologically active pharmacophore such as furan with an acid hydrazide to obtain Schiff bases as source of potential antimicrobial and anthelminitic agents. The five membered oxygen containing heterocyclic nucleus was also substituted at 5^th position to study the effect of various substituents on biological activity. It has been reported that hyrazide Schiff bases prepared from aromatic hydroxy aldehydes found significant applications in analytical and pharmaceutical industries (Nath et al., 2001; Tarafdar and Khan, 1991). Schiff bases have been of great importance due to their synthetic flexibility, selectivity and sensitivity towards the metal ions. Heterocyclic ring containing oxygen atom further imparts broad spectrum of biological activity to Schiff’s bases (Nair et al., 1983).

The title compounds, N-[{5-(substituted aryl)-furan-2-yl}-methylidene]-hydrazides (4a-f, 4g-k) Schiff bases were synthesized by condensing ketohydrazide 2 with 5-(phenyl substituted)-2-furfuraldehyde 3 in presence of sulfuric acid (Scheme 1). The compounds were characterized by IR, ¹H-NMR, Mass spectral data and elemental (C, H, N) analysis. The compounds were obtained in moderate to good yield. The IR spectra of compounds (4a-f, 4g-k) showed absorption bands for C=O of CONH at 1639-1698; NH of CONH at 1607-1648 and at 3312-3369 cm^-1. The ¹H-NMR spectra of title compounds showed the typical protons signals for N=CH and CONH groups at δ 9.37-10.14 and 9.93-10.24 ppm range, respectively. The molecular ion peak (M⁺) for all the synthesized compounds was also obtained in mass spectra and was of good intensity.

Scheme 1: Protocol for the synthesis of hydrazide-Schiff bases

Biological evaluation

Antibacterial activity

In vitro antibacterial activity of the title compounds (4a-f, 4g-k) was carried out against eight human pathogenic bacteria, four Gram positive bacterial strains; S.aureus, B. cereus, E. faecalis and S. epidermidis, and four Gram negative bacterial strains; E. coli, S. typhi, S. dysenteriae and K. pneumonia, respectively. The zone of inhibition of positive control ampicillin, against Gram positive bacteria was 22-26 mm and against Gram negative bacteria was in range of 22-27 mm. The least sensitive Gram positive and negative bacteria against ampicillin were S. epidermidis and E. coli, while E. faecalis and S. typhi were most sensitive. All the tested compounds (4a-f, 4g-k) showed good antibacterial activity against all the tested strains. An analysis of results showed that, among tested compounds, the compounds 4c, 4f, 4g, 4i, 4j and 4k which have electron withdrawing groups (4-bromo, 2,4-dinitro, 4-sulfoxy, 3-chloro, 4-carboxylic, and 4-sulfacetamido) on the phenyl ring, showed excellent antibacterial activity and found to be equipotent against B. cereus, S. dysenteriae, S. aureus, S. epidermidis and K. pneumoniae when compared with standard drug. Compounds 4a, 4b, 4d and 4h showed moderate antibacterial activity against S. aureus, B. cereus, E. faecalis, and S. epidermidis, E. coli, S. typhi, S. dysenteriae and K. pneumonia (Table II). It was interesting to note that compound 4k showed better zone of inhibition (25 mm) than the standard drug (24 mm) against S. epidermidis and also matched the standard (26 mm) in antibacterial action against K. pneumoniae. Like our compounds, Schiff bases derived from isatin derivatives and N[4-(4' chlorophenyl)thiozole-2-yl] thiosemicarbazide, have also shown to be potent antimicrobial agents (Pandeya et al., 1999). The antibacterial activity of N-(1-phenyl-2-hydroxy-2-phenylethylidine)-2',4' dinitrophenyl hydrazine was found to be at par with kanamycin at the same dose against S. aureus, B. megaterium, E. coli, S. dysenteriae, S. sonnei and P. aeruginosa (Jesmin et al., 2008). However, Siddique et al., in 2009 evaluated the antimicrobial activity of Schiff bases prepared from 4-chloro benzaldehyde and substituted hydrazides and reported them to be moderately active against Typhimusium Salmonella, Candida albicans, E. coli, B. subtilis while found to be inactive against S. aureus (Siddique et al., 2013).

Anthelmintic activity

The helminthes are the common cause of parasitic diseases in poverty stricken and developing countries with warm, moist environments with poor sanitary conditions (Sarnaim et al., 2013). Anthelmintic agents get rid of the parasitic worms by expelling them from the host body but the extensive use of these drugs have led to the development of resistance which necessitates the design and synthesis of potent and safe anthelmintic agents. Indian earthworms, P. posthuma and P. excavatus, due to their anatomical and physiological resemblance to the intestinal roundworm parasites in humanswere used for the evaluation of anthelminitic activity of the synthesized compounds.

The newly synthesized Schiff bases showed moderate to good anthelmintic activity at 2 mg/mL concentration. The results revealed that all the tested compounds were found to be effective against P. posthuma and P. excavatus, possessing significant activity in respect of mean paralyzing and mean death time. The mean paralyzing time (min) of tested compounds against P. excavatus and P. posthuma, was observed to be 9.10-20.35 and 11.6-20.6 min in comparison to 10.1 and 11.5 min shown by albendazole (Table III). The most and the least potent anthelmintic compound in terms of mean paralyzing time against P. excavatus was noted to be 4f (9.1 min) and 4h (20.4 min), while against P. posthuma, 4b and 4g had the similar spectrum of activity. The Results were comparable to that of the standard drug. The mean death time observed for albendazole against P. posthuma and P. excavatus was 17.9 and 15.7 min. Compounds 4a and 4b were found to be more potent than standard drug in causing death of nematodes, which took an average time of 15.5 and 16.3 min against P. excavatus and P. posthuma, respectively. Various condensed products of hydrazino benzthiazoles and isatin also showed the better activity than standard drug albendazole (Suresh et al., 2011). The possible mechanism of anthelmintic activity of these compounds could be due to their ability to provide two potential donor sites viz. oxygen and nitrogen atoms of ketohydrazide Schiff bases, which help in formation of hydrogen bond(s) (Venugopala and Jayashree, 2003) and thus might lead to the paralysis/death of parasitic worms.

The following is the proposed structure activity relationship (SAR) of the synthesized compounds: a) It was observed that substitution of aryl ring present at C-5 position of furfuryl ring exhibits significant antibacterial and anthelmintic activities; b) introduction of substituted aryl ring in the Schiff bases enhances antimicrobial and anthelmintic actions; c) presence of electron withdrawing groups leads to improved and broad spectrum of biological activity.

Conclusion

To sum up, the newly synthesized hydrazide Schiff bases were successfully synthesized and evaluated for their antibacterial and anthelmintic activities with significant results. The study demonstrates the antibacterial and anthelmintic potential of hydrazide Schiff bases.

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